Method of determining a number of platelets

ABSTRACT

A method of determining a measure of the number of platelets in a cell suspension containing platelets. The number of small particles in the suspension is counted. The suspension is agitated in the presence of a gas. The number of small particles in the suspension after agitation is counted. The two counts are compared to obtain a measure of the number of platelets.

FIELD OF THE INVENTION

The present invention relates to a method of analysing a sample of freecells, in particular blood cells.

BACKGROUND TO THE INVENTION

Automated block analysers which count and size blood cells, represent ahuge advance in the field of chemical medicine, but retain somedrawbacks. They are inherently incapable of differentiating like sizedparticles. While the automated count may be correct in terms of thetotal number of particles, traditional methods do not count small redcells, parasites or bits of cells as platelets. Anyone in need of goodquality platelet counts is well advised to travel to Africa or Asiawhich, having few automated blood analysers, provide better qualitycomplete blood counts because manual methods of counting avoid severalerrors which are common concomitants of automated sizing and countinginstruments. Errors in counting platelets have serious consequences forthe patient as they may result in unnecessary tests, inappropriatetreatment, or mis-diagnosis. The known causes of spurious low plateletcounts are EDTA dependent clumping, cold platelet agglutination,platelet satellitism and the presence of like sized particles which arenot platelets.

All haematology texts, laboratories and manufacturers have been aware ofthe inaccuracies of automated platelet counts for many years and advisea manual inspection of a blood film for every patient with an abnormalplatelet count, which is the practice in most haematology laboratories.Currently manufacturers are attempting to reduce errors by detectinglight refraction (since platelets are more refractile) or by detectingstains applied to platelets before they reach the sensor. The practiceof manual verification whenever the platelet measurements fall outsidethe normal range is comforting to the patient but expensive for thelaboratories. However, hitherto, there was no method of alerting labs ofthe need for manual verification for patients with high platelet countsthat erroneously fell inside the normal range. Thus automated plateletcounts are of limited value when platelet counts are low, they are ofuncertain value when the counts are normal, and are not entirely securewhen the counts are elevated (thrombocytosis).

As will be discussed below, the applicants have discovered that thereare other small particles, cell fragments, which are also not detectedand properly distinguished by existing automated blood cell analyzers.The importance of cell friability or the generation of cell fragmentshas hitherto not been recognised.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided amethod of determining a measure of the number of platelets in a cellsuspension containing platelets, the method comprising the steps of:counting the number of small particles in the suspension; agitating thesuspension in the presence of a gas; counting the number of smallparticles in the suspension after agitation; and comparing the twocounts to obtain a measure of the number of platelets.

The method of the invention differentiates platelets from other smallparticles exploiting a simple physiological property and removesspuriously low automated platelet counts. The accurate measurement ofplatelet number and function is of great consequence to a patient'shealth. Aside from bleeding to death from thrombocytopenia (insufficientplatelets), platelets are risk components of strokes, heart attacks, andinflammation, and are important elements in the growth of epithelialmalignancies and metastases.

All red cells are very sensitive to osmotic stress, and most cells aresensitive to mechanical stress. It has been found that platelets arerelatively insensitive to mechanical and osmotic stress except whenexposed to contact with air. Platelet suspensions alter their propertiesupon exposure to air, or its component gases to a greater extent if theyare simultaneously stressed. By carefully controlling the handling ofplatelets before and during testing, by eliminating or regulating asuspension's exposure to air, more accurate platelet counts can beobtained. By intentionally exposing platelets to air while subjectingthem to stress any induced change in the platelet population can berecorded. Because existing methods ignore the effect of air, they areinducing errors in their platelet counts.

The methods described in WO 97/24598, WO 97/24599 and WO 97/24601provide a way to measure the size, shape, and number of particles whilethe particles are simultaneously exposed to a variety of osmoticgradients. However, further information may be derived from this test bycombining it with the present invention. For example, by testing a wholeblood suspension under osmotic stress, and comparing the results to thesame sample after mechanical agitation in the presence of air, thecomponents of the sample population which are altered by mechanicalagitation in the presence of air can be determined. Furthermore, thetypes and proportion of each type can be revealed in a single procedure.Thus, red cells, white cells, micro-spherocytes, and bacteria areuninfluenced by mechanical agitation in the presence of air whereasplatelets alone disappear. The advantage of this method in that inaddition to explaining a way to differentiate and count platelets, itoffers existing instruments a very simple method of increasing theaccuracy of their counts.

In this application we also disclose a method of measuring cellfriability since mechanical stress induces fragmentation in those cells.When cells are counted in cell counters, neither lysis nor fragmentationnor ghosts are induced. When cells are subjected to osmotic stress theylyse and ghost or fragment or both. The addition of mechanical agitationenhances the effect and the addition of air into the agitated suspensionfurther intensifies the effect. Thus, in order to differentiate betweencell fragments and platelets, it has been found to be preferable tocompare the count of small particles before and after agitation in thepresence of air against osomolality. This process of inducing the natureof these small particles can be likened to the identification of anunknown fluid by raising its temperature; if it boils at 78° C. it isethyl alcohol, if it boils at 100° C. it is water and at 357° C. it ismercury.

In order to provide quantative measures from the present invention, itis preferable to monitor at least one of the following: A) the airquantity in contact with the suspension, B) the intensity of agitationand C) the duration of agitation, and to relate this measured value tothe difference between the two counts.

The small particles counted in the method of the invention includeplatelets, bacteria, cell fragments, and micro-spherocytes, whichtypically have a volume of 7-10 femtoliters.

According to another aspect of the present invention, a method ofanalysing a sample of free cells in vitro comprises applying a known oridentifiable quantity of stress to the sample, measuring the samplebefore and after the application of stress to provide at least onereading from which quantitative information relating to the number ofcell fragments in the sample caused by the applied stress can bedetermined, and relating this reading to the quantity of stress toprovide an indication of cell friability.

When red cells die, they lose their contents, a process termed lysis.They are then transformed into either ghost cells or fragmentsdepending, in part, on the cell's membrane properties, and in part onthe provocation. Hitherto, this mechanism has been little recognised orunderstood.

The present invention is based on the realisation that if cells, such asblood cells, and in particular red cells, are stressed in vitro therelationship between the applied stress and fragmentation to the sampleis characteristic for normal samples and for many diseases. Since somecells (platelets) produce no detectable fragments and some (red cells)produce a large number this method also provides a way of distinguishingbetween certain cell types.

Existing automated particle analysers are of limited use in detectingfragments as they cannot identify fragmentation that was cleared in vivoby increased phagocytosis, and when fragments were not phagocytosed, itcannot easily differentiate them from platelets, apoptotic bodies,micro-spherocytes, parasites and noise. There are many patients whosecells are fragmenting yet no fragments are detected by existing methodsbecause the body's physiological clearing mechanism removes fragments asfast as they are produced.

The method of the present invention has been found to be remarkably goodat identifying certain blood abnormalities because it detects patientswhose cells fragment when stressed. Because applied stress affects redblood cell fragments and platelets differently, it is possible todistinguish between the two. This has not been possible in the prior artparticle counters because platelets and red blood cells fragments areusually not measured individually. In addition, as a sample can betested before, during and after a stress has been applied, the inducedeffect provides an indication of the physiological fragmentationpotential and the physiological removal rate.

The invention can be used to identify the age of population of cellssince old cells fragment readily whereas new ones do not fragmentreadily. This may be useful in blood-banking, for instance, to destroyselectively a particular population of cells using a particular range ofstresses, thereby prolonging the life of the unit by culling the oldcells.

The applied stress can be mechanical, chemical, thermal, sonic, light,electric, electromagnetic, or any other means which induces membranestress including in vitro aging of the cell sample. Preferably, thecells are subjected to an osmotic gradient. They may also be agitated bya stirring bar, by shaking vigorously, or by any other type of stress.More than one stress mechanism can be used at the same time ifnecessary. Indeed this may be beneficial in identifying certainabnormalities.

It is, however, preferable to apply a known stress, and more preferableto apply a range of stresses, which may be decreasing or haphazard, butis preferably increasing, so as to obtain a plot of the relationshipbetween the number of red blood cell fragments and the applied stress.This range of stresses can be applied by varying duration and/orintensity, for example, by virtue of mechanically agitating the sampleincreasingly vigorously, by mechanically agitating the sample at aconstant rate over an increasing period of time, by diluting the samplewith a solution which gradually decreases in osmolality, or by anycombination of these mechanisms. The latter approach can be carried outusing the apparatus disclosed in WO 97/24529, which generates anosmolality gradient.

Characteristics providing the quantative information relating to thenumber of cell fragments include the detection of the fragmentsthemselves, an alteration in the frequency distributions of multiplecell populations, an increase in the total particle count, a change inthe concentration of intact cells, a change in the concentration ofmembrane or cytosolic parts, such as haemoglobin release, or other suchphenomena related to induced cell fragmentation.

The step of counting the number of small particles is preferably doneusing a conventional commercially available particle counter, or usingthe apparatus disclosed in WO 97/24600. In both cases, the blood sampleis caused to flow through a sensor, typically an aperture, where itssize is detected optically, acoustically, thermally, electronically orby other means. In an impedance sensor, the response of the electricalfield to the passage of the cells is recorded as a series of voltagepulses, the amplitude of each pulse being a function of cell fragmentsize and frequency.

In the absence of applied stress, existing instruments are unable todistinguish between platelets and, for instance, red cell fragments. Aseach different cell type has its own biochemical individuality and itsown sensitivity to fragmentation, it is possible to use its response asan identifying property, identifying the cell type, sensitivity andpathology. For instance, platelets which are-similar in size tofragments, are differentiated from fragments as they respond differentlywhen stressed. The differentiation and identification of fragments,platelets, ghost cells and other cell parts may be facilitated by usingstains and dyes which selectively dye cell lines, or bond onto specificcell parts, for instance the inside and outside of the cell membrane oronly stain the platelets. When a stress has been applied to a bloodsample, as for example, in WO 97/24598, WO 97/24599 and WO 97/24601, itwas performed to obtain accurate volume and other cell measurements byforcing cells to a known shape. The method disclosed herein inducescells to fragment and measures the result of fragmentation. This may beenhanced by, for instance, eliminating all particles except for thefragments of interest by setting the upper and lower threshold voltagesto the size range of fragments, by facilitating fragmentation byapplying heparin or other suitable chemicals in the chemical preparationof the sample and/or by allowing sufficient time for fragmentation toproceed. The induction, detection and quantification of fragmentsenhances the measures produced by WO 97/24598, WO 97/24599 and WO97/24601 and other measures which may be altered by induced or existingfragments.

When using an electrical particle counter, the thresholds should be setlow enough to detect cells down to a volume of almost 0 femtoliters. Athreshold voltage of 0.08 mV has been found to work well with theexisting apparatus. Other populations of particles may be eliminatedelectronically, digitally, mechanically, or by other means.

The commercially available particle counters and the device disclosed inWO 97/24600 can be used to provide adequate readings for the presentinvention. However, more accurate readings can be obtained if the sizeof the aperture in either device is reduced so that the ratio of thecross section of the aperture to the mean cross section of the red bloodcell fragments or platelets is substantially 4:1.

In conventional blood cell analysis techniques, it is usual to treat thesample with an anticoagulant, such as EDTA, prior to analysis. However,it tends to inhibit fragmentation. It has therefore been foundpreferable not to treat the sample with an anticoagulant or to use ananticoagulant which does not inhibit or promote fragmentation, such asheparin.

BRIEF DESCRIPTION OF THE DRAWINGS

Examples of the present invention will now be described in detail withreference to the accompanying drawings, in which:

FIGS. 1 to 5 are frequency distributions indicating the profile of cellsize measurement against increasing osmotic and mechanical stress for anumber of cell samples.

DETAILED DESCRIPTION

Examples of results obtained using the method according to the presentinvention, will now be described with reference to FIGS. 1 to 5 of theaccompanying drawings, which are frequency distributions indicating theprofile of the cell size measurement against increasing stress for asample of cells tested using the apparatus disclosed in WO 97/24529 andthe electrode disclosed in WO 97/24600.

Such frequency distributions have previously been used, for example inWO 97/24598, WO 97/24599, and WO 97/24601, for example to provideindications of the cell permeability, osmolality and cell shaperespectively of normal unfragmented red blood cells.

The samples tested were treated with heparin, mechanically agitated bystirring or shaking in air, then subjected to an osmotic gradient usingthe apparatus disclosed in WO 97/24529.

FIG. 1 is a blood sample from a healthy person. As the stress isincreased from point A onwards, the cells begin to swell resulting inthe gradual increase in mean cell size. At a certain osmolality the cellsize can increase no more, and upon a further increase-in stress, thecells evacuate their contents to become “ghost cells” as shown at regionB. These characteristics of cells yield diagnostic information which isuseful alongside the techniques of the present invention. However, thepresent invention is unconcerned with these characteristics and isindependent of them, but is instead concerned with the cells whichappear along the baseline of the graph of FIG. 1. The nature of thesesmall baseline cells can be differentiated by their distribution againstosmotic stress, their distribution against mechanical stress and/ortheir response to contact with air. Platelets are uninfluenced byosmotic stress within the range tested (300−100 mOsm/Kg). However,fragments are strongly influenced by osmotic stress. They rarely appearwithout the provocation of lysis and become more numerous as lysisincreases. Thus fragments vary inversely with osmolality. Mechanicalstress has opposing effects on platelets and fragments, an influencethat is intensified by concomitant exposure to air. It provokes theappearance of fragments but it induces the lysis of platelets renderingthem invisible.

FIGS. 2A-F demonstrate that platelets are rendered invisible withprogressive agitation in air (with respective agitation periods of 0, 1,2, 6, 10 and 15 seconds). The signal along the base line in FIG. 2A isattributable to the presence of platelets since their frequency isapproximately the same at all osmolalites, and since even 2 seconds ofagitation in air induces almost complete disappearance (see FIG. 2C).The phenomenon cannot be attributed to platelet attachment to the redcells or white cells (satelitism) since FIGS. 3A-F shows the samephenomenon in a pure platelet suspension when no other cells types arepresent.

FIGS. 4A-E demonstrate the effect of mechanical agitation in air withtime. As the agitation period proceeds going from 4A down to 4E thenumber of fragments generated increases. At the same time, platelets arerapidly made invisible (although it is difficult to see this in theseparticular figures). FIGS. 5A-D demonstrate the platelet reduction in awhole blood sample on shaking with air. Such frequency distributionshave previously been used, for example, in WO 97/24598, WO 97/24599, WO97/24601.

The preferred method comprises measuring platelets, using existingautomated particle counters, mixing and testing the sample in theabsence of air. If some air is unavoidable, the surface area of thesamples meniscus should be minimized to limit the effect on the sampleor alternatively a gas which has no effect on the platelets should beused. This single measurement will provide an accurate count for allsamples which do not contain particles of a similar size to theplatelets. Repeat measurements are taken after controlled agitation withair to render the platelets invisible to an automated analyser. Thedifference between the population from the two measurements provides ameasure of the number of platelets, their age and physiologicalproperties. In a whole blood sample, only the platelets will disappear.For instance, if the cell counts before and after agitation with air donot differ, then there are no platelets in the suspension.

We have measured platelets under a variety of experimental conditions:in pure platelet suspensions, in the presence of all other blood cellelements, in containers of glass, polystyrene and polypropylene, afterexposure to varying surface areas in a thin film and by shaking withvarying quantities of glass beads, after exposure to varying osmotic,mechanical, thermal, and sonic stress, and after mechanical agitationwith air and nitrogen.

By measuring the platelet count against all these stresses, we havefound conditions which render platelets electronically invisible, whileother types of cells are unaltered or transformed but remain visible toautomated analysers. Moreover the speed and characteristic of thedisappearance is an indication of platelet health and function.

In all laboratories, prior to any analysis on any blood sample, thesample is first shaken to mix the cell types randomly with respect toeach other and with the plasma. The agitation is continued during theprocess of analysis in automated instruments, and the cells are exposedto agitation in the presence of a variable amount of air, bothdeliberately and accidentally. Indeed, many instruments use air bubblesas a way to separate each bolus of blood or clean the tubes andinstrument conduits.

Shaking with glass beads has a small effect on the platelet count andmay therefore be preferable to shaking with air or some other gas forinitial sample mixing when the aim is to avoid platelet disappearance.The mechanical effect of shaking was further differentiated from theeffect of air admixture by continuously withdrawing aliquots from aconical flask when it contained no air (being full of the bloodsuspension) until it was empty containing nothing but air. All the whilethe blood suspension was continuously agitated with a stirring bar. Theplatelet count remained constant so long as the flask was full, but oncethe sample/air ratio fell below a half, the platelets began to disappearand did so in direct proportion to the volume of air in the flask. Inthe absence of air, the platelets do not disappear appreciably withinseconds as they do in the presence of air. Thus counting the plateletsagainst duration or intensity of agitation with air (or its components),will induce them to diminish or completely disappear but bacteria,fragments or any other element that may be found in the blood withneither diminish nor disappear.

The method is useful in the following:

1. IMPROVED DISEASE DIAGNOSIS. More accurate platelet counts will leadto better disease diagnosis and prognosis.

2. EXISTING METHODS OF BLOOD TESTING. By using an inert gas, oreliminating air from the mixing, automated instruments will improve theaccuracy and repeatability of platelet counting and sizing.

3. A NEW MEASURE OF PLATELET HEALTH. By using this method, newinformation about platelet health is derived from their sensitivity toair and their rate of disappearance and reappearance.

4. BLOOD COLLECTION. Currently Vacutainers (trade mark) (partial vacuumglass specimen tube) contain air, and more is sometimes admitted whenthe sample is smaller than required.

What is claimed is:
 1. A method of determining a measure of the numberof platelets in a cell suspension containing platelets, the methodcomprising the steps of: counting the number of small particles in thesuspension; agitating the suspension in the presence of a gas; countingthe number of small particles in the suspension after agitation; and,comparing the two counts to obtain a measure of the number of platelets.2. A method according to claim 1, in which the gas is air, or one ormore of its constituent gases.
 3. A method according to claim 1, inwhich one or more aliquots of the suspension are passed through aparticle counter before the suspension is agitated to obtain a firstcount of the number of small particles and subsequently one or moreother aliquots of the suspension are passed through a particle counterafter agitation to obtain a second count of the number of smallparticles in the sample.
 4. A method according to claim 1, in which thecell suspension is isotonic.
 5. A method according to claim 1, in whichsmall particles having a volume of at least 7 femtoliters are detectedto obtain a count of the number of small particles in the suspension. 6.A method according to claim 1, in which the suspension is subjected to aseries of alterations in osmolality and a count of the number of smallparticles in the suspension obtained at a number of differentosmolalities.
 7. A method according to claim 1, in which at least one ofthe air quantity in contact with the suspension, the intensity ofagitation, and the duration of agitation, is measured.